G protein-coupled receptors (GPCRs) are the targets of many prescription and illicit drugs and are involved in mediating drug dependent behaviors. The ability of GPCRs to maintain or halt signaling critically depends on how they traffic after ligand-induced endocytosis, but this process remains poorly understood. The protein complex classically involved in retrograde transport of cargos from endosomes to the trans-Golgi network, Retromer, was recently discovered to also be utilized in the recycling of several GPCRs from endosomes directly to the plasma membrane, a novel function for this complex. Retromer is thought to act as a coat-complex, forming membranous tubular structures which protrude from endosomes to pinch off into vesicles that traffic to different destinations. I hypothesize that the Retromer complex is able to physically and biochemically distinguish cargos in order to transport them to distinct destinations. I propose to rigorously test this hypothesis using two GPCR cargos that are implicated in drug-dependent behavior and that rapidly recycle: 1) the mu-type opioid receptor (MOR), the target for opioid peptides and drugs, and 2) the beta-2-adrenergic receptor (beta-2AR), a catecholamine receptor implicated in reward-based learning. As a comparison, the GPCR-like Wnt transport receptor, Wntless (Wls), will be used model cargo for the retrograde transport pathway. Interestingly, Wls has also been identified as an interaction partner for MOR, so I will also investigate how the presence and activation of MOR affects the trafficking of Wls. The specific aims of my project are to: 1) determine the physical branch-point of Retromer-dependent recycling and retrograde transport cargos in model cultured cells, 2) determine if proteins involved in Retromer-dependent recycling and retrograde transport are specific for and sufficient to control cargo trafficking destination and define their mechanisms of action in model cultured cells and, 3) determine if there are differences in Retromer-dependent trafficking mechanisms found in model cultured cells in primary striatal medium spiny neurons, a cell type essential to driving drug-dependent behavior. These aims will be addressed using a variety of techniques including microscopy, fluorescence flow cytometry, and biochemistry. The proposed project addresses the problem of specificity of GPCR regulation by endocytic membrane trafficking, which is fundamental to our understanding of the cellular basis of drug abuse and addiction.